Mechanism of growth inhibition of prostate cancer xenografts by valproic acid

Abstract

Valproic Acid (VPA), a histone deacetylase inhibitor, has been demonstrated to cause a marked decrease in proliferation of prostate cancer (PCa) cells in vitro and a significant reduction in tumor volume in vivo. The goal of this study is to better understand the VPA-induced growth inhibition in vivo, by studying expression of various markers in PCa xenografts.

Methods: For in vitro experiments, PCa cells were treated with 0, 0.6, and 1.2 mM VPA for 14 days. For in vivo models, experimental animals received 0.4% VPA in drinking water for 35 days. Tissue microarray was generated using cell pellets and excised xenografts.

Results: VPA treatment causes cell cycle arrest in PCa cells in vivo, as determined by increase in p21 and p27 and decrease in cyclin D1 expression. Increased expression of cytokeratin18 was also seen in xenografts. LNCaP xenografts in treated animals had reduced androgen receptor (AR) expression. While decreased proliferation was found in vitro, increase in apoptosis was found to be the reason for decreased tumor growth in vivo. Also, an anti-angiogenic effect was observed after VPA treatment.

Conclusion: VPA inhibits tumor growth by multiple mechanisms including cell cycle arrest, induction of differentiation, and inhibition of growth of tumor vasculature.

Figure 1

Animals with prostate cancer xenografts were randomized into control and treatment arms. Animals in treatment arm received 0.4% w/v Valproic Acid (VPA) in drinking water. Animals were treated for 35 days before excision of tumors. Tissue Microarrays (TMAs) of xenografts were created and expression of various markers was studied. Representative images of xenograft sections from control and VPA-treated groups; LNCaP, C4-2, and DU-145 (scanned at 20x magnification using the APERIO imaging system) are shown. (a) Immunohistochemistry (IHC) staining for acetylated histone 3. (b) Staining of cell cycle regulators (p21, p27, cyclin D1). (c) Staining for differentiation markers (Cytokeratin 18 (CK18), androgen receptor (AR)). (d) Staining for markers of proliferation (Ki-67), apoptosis (TUNEL), and angiogenesis (mean vascular density, MVD).

Figure 2

Cell lines (LNCaP, C4-2) were treated with 0, 0.6, and 1.2 mM of VPA for 14 days. TMA were created from cell pellets followed by IHC staining for expression of markers. Representative images of p21, p27, CK18, AR, and Ki-67 staining are shown (scanned at 20x magnification using the APERIO imaging system).